Monkeypox virus and variola virus share about 95% homology, and cause similar diseases in humans. Yet one of the major determinants of orthopoxvirus virulence, the E3L gene, differs significantly between these two closely related viruses. The monkeypox virus homologue of the variola virus (and vaccinia virus) E3L gene, which is an important vaccinia virus innate immune evasion gene, is partially deleted. Since vaccinia virus, when expressing a similarly deleted E3L is attenuated in experimental animals, it is important to understand the role of this variant monkeypox virus innate immune evasion gene in replication and immune evasion of monkeypox virus. The research in this application will characterize the altered genes in monkeypox virus that are compensating for the partial deletion of the monkeypox virus E3L homologue. As such the work described in this application will provide important information on how monkeypox virus causes disease in animals and in humans. Since monkeypox virus is considered to be a re- emerging human public health concern, this research may provide the basis for development of better vaccines for protection against monkeypox virus infection in humans, and for the development of better treatments for people who do become infected with monkeypox virus.